Fully Reduced HMGB1 Enhances Nonunion Fracture Healing in Diabetic Rat Model

Abstract

Category: Basic Sciences/Biologics; Diabetes Introduction/Purpose: Diabetes mellitus is a chronic metabolic disease that leads to serious complications including bone health and wound healing. Bone fractures commonly occur in diabetic patients, and are difficultly to heal due to prolonged inflammation, which leads to a higher risk of non-union. Fully reduced high mobility group box1 (frHMGB1) can play an important role in skeletal regeneration; however, the activity can be inhibited by Metformin; which is commonly used for the treatment of diabetes. The effect of frHMGB1 and Metformin on the bone fracture healing is largely unknown. In this study, we tested the hypothesis that frHMGB1 enhances healing in a nonunion bone fracture and Metformin inhibits the healing using a diabetic rat model. Methods: Diabetic fracture rat model was created by injection of streptozotocin into total 32 Sprague Dawley rats and confirmed by blood glucose levels.An incision was performed, tibia bones were fractured, followed by the muscle and skin wound closure.The rats were divided into four groups with 8 rats per group(Fig 1): Group-1 had no additional treatment;Group-2 were injected Metformin (IP, 160 mg/kg) daily;Group-3 were injected frHMGB1 into wound area (250 µg/kg) weekly;Group-4 were injected Metformin (IP, 160 mg/kg) daily and frHMGB1 into wound area (250 µg/kg) weekly. The rats were monitored postoperatively and sacrificed on postoperative day-28 and day-90. The blood samples were collected for HMGB1 & IL-1β.Tibia samples were also collected and examined by gross inspection, micro-CT images and histological analysis. Data was analyzed by One-way ANOVA followed by Fisher’s Least significance difference test. A p-value less than 0.05 was considered to be significantly different between the groups. Results: Metformin with frHMGB1 significantly decreased HMGB1 levels and decreased the enhanced expression of IL-1β compared to frHMGB1 alone. Micro-CT images showed completely healed at day-90 post-surgery in frHMGB1 rats while Metformin inhibited frHMGB1 enhanced bone fracture healing as evidenced by the gap in the fractured bone area(Fig 2). Histology analysis indicated that frHMGB1 enhanced fractured bone healing as evidenced by high density of the cells was found in the bone fracture area (Fig. 3). Safranin O and fast green staining along with Masson’s Trichrome staining confirmed the frHMGB1 local injection promoted fractured bone healing (Fig. 4) as evidenced by high quality bone tissue formation with collagen type I (red staining) and collagen type III (blue staining) in the fractured bone wound area. Conclusion: Fully reduced HMGB1 treatment enhanced healing in a nonunion diabetic rat model. The fracture site had high density of cells, quality bone tissue, and enhanced production of collagen I and III. However, Metformin inhibited frHMGB1-induced healing enhancement. In diabetic rats, nonunion bone fracture was present with evidence of inflammation showing elevated levels of HMGB1 and IL-1β.FrHMGB1 treatment enhanced healing of nonunion bone fracture by promoting cell proliferation and migration, limited HMGB1 concentration, increased collagen production. Although Metformin injection slowed down the bone fracture healing by inhibiting frHMGB1activity, it also blocked HMGB1 release and reduced the inflammation caused by HMGB1.